Calcium phosphate is the name given to a family of minerals containing calcium ions (Ca2+), together with orthophosphates (PO43−), metaphosphates or pyrophosphates (P2O74−) and hydrogen or hydroxide ions. One of the naturally occurring forms of calcium phosphate present in bones and tooth enamel is biological hydroxyapatite (HA) which has the approximate formula Ca5(PO4)3(OH), usually written Ca10(PO4)6(OH)2. In the biological field, synthetic crystalline hydroxyapatite is used in tissue engineering, primarily as a filler material for repairing bones and teeth. Nanoparticles of hydroxyapatite have also been proposed as carriers for drugs and have been employed in imaging techniques.
Hydroxyapatite can be prepared in a precipitation reaction of calcium and dibasic phosphate salts in neutral or basic solution and has as its final product crystalline hydroxyapatite. However, during precipitation, a structurally and chemically distinct precursor phase is formed which is amorphous to X-ray diffraction, known as amorphous calcium phosphate (ACP).
Chemical analysis of the precursor phase indicates this non crystalline phase is a hydratea calcium phosphate. As with other amorphous materials, several formulae have been proposed for ACP, such as Ca9(PO4)6.
Hydroxyapatites have been used as carriers for biomolecules, in particular for DNA transfaction, drug delivery, and in orthopaedics and dentistry. By way of example, Chowdhury et al. have investigated delivery of DNA to mammalian cells in culture by precipitating DNA with calcium phosphate in the form of crystalline hydroxyapatite (see Gene, 341: 77-82, 2004; J. Controlled Release, 116(2): e68-e69, 2006; Analytical Biochemistry, 328: 96-97, 2004; US 2007/0077306). These experiments included using Mg2+ as an agent to inhibit the growth of particles of precipitated hydroxyapatite and DNA to avoid a loss of transfection efficiency associated with an increase in particle size. However, while Mg2+ was incorporated into the apatite particles precipitated with DNA, the particles remained, crystalline, Dasgupta et al. reported the use of Zn- and Mg-doped hydroxyapatite nanoparticles as controlled release carriers for bovine serum, albumin (Langmuir, 26(7): 4958-4964, 2010). However, as with the studies reported by Chowdhury et al., the doped, hydroxyapatite materials produced retained a clear degree of crystallinity in common with unmodified hydroxyapatite.
During synthesis, ACP rapidly converts (in the presence of water) to microcrystalline hydroxyapatite and the lifetime of the metastable ACP in aqueous solution has been reported to be a function of the presence of certain macromolecules and interfering ions, pH, viscosity, ionic strength and temperature. Boskey & Posner (1973, 1974) studied the kinetics of the conversion and found that substitution of Ca ions in ACP by Mg ions leads to greater stability of the amorphous state, lessening its tendency to convert through to more crystalline phases such as hydroxyapatite. They showed that at a ratio of at least 1:25 (Mg:Ca), an amorphous magnesium calcium phosphate phase is produced that, as a dry powder, remains stable over time.
While the synthesis of ACP has been reported, only very limited applications of this material have been proposed in the fields of dentistry and tissue engineering as a structural material for use in repairing bones and teeth and as a scaffold for tissue engineering. By way of example, Zhao et al. (Chemistry Central Journal, 5: 40-47, 2011) describe the use of amorphous calcium phosphate in dentistry as a composite for re-mineralising and repairing teeth. They report that in the presence of other ions and under in vivo conditions, ACP may persist for appreciable periods due to kinetic stabilization in the presence of Mg2+, F−, carbonate, pyrophosphate, diphosphonates, or polyphosphorylated metabolites or nucleotides, preventing the transformation of synthetic ACP to hydroxyapatite. Li & Weng (J. Mater. Sci.: Mater. Med., 18: 2303-2308, 2007) reported the synthesis of amorphous calcium phosphates (ACP) and were using poly(ethylene glycol) as stabilizing additive at low temperature. They found that ACP could be stabilized by poly(ethylene glycol) in the mother solution for more than 18 hours at 5° C. with 4 w.t. % poly(ethylene glycol) in ACP powders and suggested that ACP might be used as biodegradable scaffold for tissue engineering.
Peyer's patches are lymphoid follicles that perform critical immune sensing and surveillance functions in the gastrointestinal tract. The region beneath the Peyer's patch epithelium is referred to as the sub-epithelial dome (SED) and is enriched, with antigen presenting cells. Whole bacteria and similar sized microparticles of the gut lumen can be directly phagocytosed by specialised SED dendritic cells which migrate upwards and extend dendrites through the follicle associated epithelium. For the surveillance of soluble molecules and smaller particles the epithelium contains distinctive microfold (M) cells that appear to sample the lumen directly and transport the sampled material to underlying immune cells. Exactly how this occurs and how antigen, for example, is not degraded en route is not understood.
It is also unclear why Peyer's patch M cells avidly sample non-biological nanoparticles of ˜20-250 nm diameter from the gut lumen. Nonetheless that it occurs is well demonstrated in cellular and animal models and also for humans with normal day-to-day exposure to nanoparticles from processed foods, pharmaceuticals and toothpaste.